A chemokine is a cytokine that modulates migration and cell function of various cells. Functional abnormality of a chemokine and its receptor causes various diseases such as autoimmune disease, acute and chronic inflammation, and cancer. Up to now, drugs that control the activities of a chemokine and its receptor have been developed and clinically applied, but it is hard to say that the problems are satisfactorily solved.
In order that a specific chemokine elicits its activity such as modulation of cell migration or cell function, it is necessary that the chemokine binds to a chemokine-selective cell membrane receptor. About 20 kinds of chemokine receptors have been found, and any of these chemokine receptors is a seven transmembrane type protein (GPCR) that binds to a trimeric G protein. When a chemokine binds to a receptor, it induces dissociation of the Gα unit of a trimeric G protein. As a result, it causes a increase in intracellular Ca concentration, or activates phosphatidylinositol 3-kinase (PI3K), or the small Rho GTPases pathway, or other pathways to lead expression of function. Chemokine receptors are activated by a relatively selective chemokine, but they share very similar primary structures of protein and intracellular activation mechanisms. Therefore, it is not easy to selectively interfere with the function of a specific chemokine receptor. Eliciting of function of respective chemokine and chemokine receptor in physiological and pathological conditions is controlled by each protein expressed during a specific period of time (during inflammation) in a specific cell or tissue (Non-Patent Document 1).
Human CC motif receptor 7 (CC MOTIF, RECEPTOR 7; also called: EBI1, CMKBR7; hereinafter referred to as “CCR7”) is originally found as a GPCR that is expressed in a lymphocyte-selective manner by EPSTEIN-BARR virus infection (Non-Patent Document 2). Afterwards, CCR7 was proved to be a selective chemokine receptor for CCL19 (also called ELC) and CCL21 (also called SLC, EXODUS 2). Under physiological conditions, CCR7 is expressed in cells such as CD4-positive T cells (Th1, Th2, Treg cells), mature dendritic cells, and B cells relatively selectively. It is known that such cells are led to a lesion such as an inflamed site via CCR7 to increase the inflammatory reaction and the immunization reaction. Abnormal activation of CCR7 causes various diseases such as autoimmune disease, fibrosis following acute and chronic inflammation, and cancer metastasis.
The amino acid sequence of human CCR7 and the nucleotide sequence of human CCR7 gene have been already known (for example, GenBank: EAW60669.1).
Inflammation is a defense reaction against injury to tissue and infection. Following an increase in expression of inflammatory molecules (chemokines and cytokines) in response to various inflammatory stimulations, invasion of neutrophil and monocyte is promoted. As the inflammatory reaction further increases, T and B lymphocytes flow in and the pathosis becomes chronic. On the other hand, in a resolution stage of inflammation, apoptosis of excess leukocytes and phagocytosis by tissue macrophages occur, and repair of the injured tissue by interstitial cells (e.g., fibroblast) is observed.
Excessive proliferation and differentiation of interstitial cells are deeply related with exacerbation of various fibrosis (hepatic fibrosis, renal fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis, and other fibrous diseases). Chronic pulmonary fibrosis is caused by formation of scars throughout the lungs, and is a poor-prognosis disease. Pirfenidone, corticosteroids (for example, prednisone) having an antifibrotic activity and/or other drugs that suppress the immune system in the body are prescribed for suppressing the process leading to fibrosis. However, at present, it is hard to say that a satisfactory therapeutic outcome is achieved.
On the other hand, in recent years, the molecular mechanism of fibrosis in the repair process after inflammatory reaction is being clarified. In such a repair mechanism, local resting fibroblasts migrate to the injured region and generate an extracellular matrix protein to promote contraction of the injury and fibrogenesis (scar). Also it has been suggested that circulating fibroblast precursor cells and fibroblasts (they are present in blood) migrate to the site of injury or fibrogenesis, where they differentiate to mediate repair of tissues or other fibrous reactions.
Fibroblast precursor cells in blood differentiate from a CD14+ peripheral blood mononuclear cell group invading the inflamed site, into stromal cell-like cells (collagen type I and type III and fibronectin) locally (Non-Patent Document 3). These cells secrete an inflammatory cytokines, and also secrete an extracellular matrix proteins and other cytokines which may lead fibrogenesis. Although it is expected that fibrosis can be minimized if excess invasion of the fibroblast precursor cells in blood into the inflamed site can be selectively suppressed, such a measure has not reached clinical application.
Recently, the aforementioned CCR7 has attracted attention as a chemokine receptor that is expressed in a fibroblast precursor cell (Patent Document 1). It has been demonstrated that fibrosis is not developed even when such stimulation that leads development of pulmonary fibrosis or renal fibrosis is added to a CCR7-deficient transgenic animal (Non-Patent Document 4). Accordingly, it is expected that various fibrosis can be suppressed if the function of CCR7 can be inhibited by a low molecular-weight compound, monoclonal antibody, RNAi or the like. However, since chemokine receptors share very similar primary structures of protein and intracellular activation mechanisms, a substance capable of inhibiting CCR7 more selectively is desired. Although substances (monoclonal antibody or RNAi) that inhibit the function of CCR7 have been studied, there is still no case that has reached clinical application.
On the other hand, in a cancer therapy, it is important to suppress proliferation of primary cancer and to prevent recurrence accompanying distant metastasis. In addition to conventional surgical therapies and chemical therapies, molecular target drugs (for example, kinase inhibitor) and antibody pharmaceutical therapies have improved therapeutic outcomes. However, recurrent cancer accompanied by distant metastasis is poor in prognosis, so that development of a novel therapeutic drug is desired. As a mechanism of distal metastasis, the primary cancer travels blood vessels or travels lymphoid tissues. Although an extracellular matrix protease inhibitor (MMP inhibitor) has been developed as a metastasis preventive drug, there is still no case that has reached clinical application.
Various studies have revealed that CCR7 is expressed in various tumor cells such as B cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer cell and malignant mammary tumor. Further it is becoming clear that CCR7 plays a role in lymph node metastasis of various cancers such as gastric cancer, melanoma, non-small cell lung cancer and T cell leukemia cell (Non-Patent Document 1). Since CCL19 and CCL21 which are ligands for CCR7 are highly expressed in lymph nodes, it is expected that selective inhibition of CCR7 function will suppress lymphatic metastasis of cancer cells.
A principal mechanism of action of an antibody drug against a membrane protein (receptor) is generally such that a cell expressing the protein is recognized by an antibody, and then removed based on complement-dependent cell lysis action (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). However, CDC and ADCC are associated with activation of inflammatory cells such as macrophages, and are not necessarily appropriate for therapy of fibrosis. Therefore, when a monoclonal antibody capable of selectively inhibiting CCR7 is applied in therapy of fibrosis, it is preferable that the monoclonal antibody is a functional antibody capable of inhibiting CCR7 without relying on CDC or ADCC. That is, an antibody that selectively interferes with CCL19- or CCL21-dependent intracellular signal transduction of CCR7 is desired. However, it is generally difficult to acquire a functional antibody against GPCR efficiently.
Patent Document 1: JP 2009-528977 T
Non-Patent Document 1: Viola A, Luster A D “Chemokines and their receptors: drug targets in immunity and inflammation”, Annu Rev Pharmacol Toxicol. 2008; 48:171-97
Non-Patent Document 2: Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G., Kieff, E., “Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors”, J. Virol. 67: 2209-2220, 1993.
Non-Patent Document 3: Pilling D, Fan T, Huang D, Kaul B, Gomer R H. “Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts”, PLoS One. 2009 Oct. 16; 4(10):e7475
Non-Patent Document 4: Wada T, Sakai N, Matsushima K, Kaneko S. “Fibrocytes: a new insight into kidney fibrosis”, Kidney Int. 2007 Aug.; 72(3):269-73.